Intellectual merit: At the cellular level, learning and memory are governed by changes in the efficacy of synaptic transmission and in particular, by the dynamic regulation of neuronal transmitter release. Neurotransmitters are packaged into synaptic vesicles that dock at the synaptic membrane, undergo a series of preparatory steps, open a pore, and fuse with the synaptic membrane, resulting in neurotransmitter release into the synaptic cleft. This process is very dynamic, plastic, and highly regulated. Although molecular components of docking and fusion have been identified, it is not yet understood how they interact to regulate the dynamics of docking, pore opening, and fusion. In particular, little is known about the detailed mechanics of protein interactions that regulate synaptic vesicle fusion. The present application will focus on this critical question by combining modeling and experimentation to investigate the molecular machinery that regulates synaptic vesicle docking and fusion. Vesicles tightly dock at the plasma membrane via a specialized protein complex (SNARE), which is thought to provide the necessary force to overcome inter-membrane repulsion and thus mediate vesicle fusion. Stimulus evoked fusion is triggered by an influx of Ca2+ ions that interact with a vesicle protein, synaptotagmin (Syt), which is tightly coupled with the SNARE complex. Fusion pore opening is thought to be controlled by the interaction of Syt and a small protein complexin (Cpx) with the SNARE complex. Although molecular interactions of these proteins have been studied with biochemical and molecular biology tools, there is still a lack of understanding of how these proteins interact dynamically and how the forces of the protein fusion machinery counterbalance forces generated within the synaptic and vesicle membranes. To elucidate these mechanisms, we propose to build a molecular model of the fusion machinery and to perform computer simulations of the dynamics of the fusion complex. To understand the interactions between the vesicle, synaptic membrane and the protein fusion machinery, we will develop a coarse grain model of membrane/vesicle dynamics and integrate it with the atomic model of the fusion protein complex. To validate the model, we will simulate the effect of single point mutations in the fusion complex on the release kinetics and test our predictions experimentally. The experiments will be performed at Drosophila neuromuscular junctions (NMJ), a model system ideally amendable to genetic manipulations. To test the predictions of the model, we will combine electrophysiology and optical fluorescent microscopy to assess release kinetics in NMJs where the fusion machinery is modified by point mutations with computationally predicted effects on membrane fusion. This research will be performed by a multidisciplinary team that includes experts in molecular modeling (Dr. Jagota), membrane mechanics and dynamics (Dr. Hui), synaptic physiology (Dr. Bykhovskaia) and Drosophila neurobiology (Dr. Littleton). An attack on this problem by a collaborative team with balanced representation of all its aspects will lead to new, detailed and quantitative, understanding of the regulated synaptic vesicle fusion process. Broader impact: Universidad Central del Caribe (UCC) is a Hispanic serving institution in Puerto Rico (U.S. Commonwealth). The proposed project will allow the UCC to develop tight links with highly regarded mainland institutions and will thus create training and employment opportunities for students with diverse backgrounds. The PI, Dr. Bykhovskaia, directs the Specialized Neuroscience Research Program (SNRP) at UCC (funded by NIH), that has a goal of raising research standards in institutions with a predominant enrollment of underrepresented minorities. Thus, the proposed project will involve underrepresented B.S., M.S., Ph.D., and M.D. students in biomedical research. Furthermore, Dr. Jagota directs the undergraduate and graduate Bioengineering programs at Lehigh University, an institution with a balanced emphasis on research, teaching and training. The proposed research will be performed by graduate students, and will actively involve undergraduate students through research for credit and summer opportunities. This research will be incorporated into the undergraduate bioengineering curriculum through a course on Biomolecular and Cellular Mechanics, developed by Dr. Jagota at Lehigh University. It will be tightly integrated with other activities, including student exchange and transdisciplinary seminars, and thus will promote integration of research and training across diverse intellectual and ethnic backgrounds.